Cerium oxide nanoparticles, methods of making and using the same

Abstract

The application discloses cerium oxide nanoparticles, a preparation method and application thereof, and belongs to the field of antibacterial materials of medical devices. 3+ The relative proportion of oxygen vacancies and lattice oxygen in the cerium oxide nanoparticles is 2.0-3.0. The application is applied to the antibacterial aspect of catheters, solves the problem that existing antibacterial materials cannot meet the antibacterial performance and biocompatibility requirements of catheters, and has the characteristics of high sterilization efficiency, low cost, high biological safety and simple preparation method.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial materials for medical devices, and particularly relates to a cerium oxide nanoparticle, its preparation method and application. Background Technology

[0002] Urinary catheters are widely used in clinical practice. However, the plastic surface of urinary catheters has poor hydrophilicity, making it easy for adhesins and extracellular polysaccharides secreted by microorganisms to irreversibly adhere to their surface. This leads to the formation of organized bacterial aggregates, resulting in biofilm formation and subsequent bacterial adhesion and infection. To improve the antibacterial properties of urinary catheters, many existing catheters use a silver-PTFE composite antibacterial coating. However, the use of silver-PTFE composite antibacterial coatings for urinary catheters still faces challenges such as high cost, complex experiments, and the non-stick properties of PTFE, making it difficult for the coating to adhere to the catheter surface.

[0003] Chinese patent CN114716817A discloses a cerium dioxide nanorod hybrid multifunctional hydrogel, its preparation method, and its application. Cerium dioxide nanorods are prepared by a hydrothermal method using a cerium source and an alkali source, and are formulated into a cerium dioxide nanorod dispersion with a mass concentration of 2.5-5%. The hydrogel is prepared by a Schiff base reaction of an aqueous solution of oxidized dextran, a cerium dioxide nanorod dispersion, an aqueous solution of polylysine, and an aqueous solution of 3,3'-dithiobis(propionylhydrazine). It is applied in a drug for treating diabetic skin lesions.

[0004] The aforementioned patent discloses an antibacterial material that can be used to treat skin injuries, but it still has the problem of not meeting the requirements for antibacterial performance and biocompatibility of urinary catheters. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the technical problem to be solved by this invention is to overcome the fact that existing antibacterial materials cannot meet the requirements of antibacterial performance and biocompatibility for urinary catheters. This invention proposes a cerium oxide nanoparticle with high bactericidal efficiency, low cost, high biosafety, and simple preparation method, as well as its preparation method and application.

[0006] To solve the aforementioned technical problem, the technical solution adopted by the present invention is as follows:

[0007] This invention provides, in one aspect, cerium oxide nanoparticles comprising cerium oxide and Chlorella vulgaris, wherein the Chlorella vulgaris is adsorbed onto the surface of the cerium oxide, and the cerium oxide contains Ce. 3+ The proportion is 20-25%, and the relative ratio of oxygen vacancies to lattice oxygen in the cerium oxide nanoparticles is 2.0-3.0.

[0008] Preferably, the cerium oxide nanoparticles are spherical with a diameter of 6-7 nm and a BET of 70-80 nm. 2 / g.

[0009] Another aspect of the present invention provides a method for preparing cerium oxide nanoparticles according to any of the above technical solutions, comprising: taking a culture medium of Chlorella growing in the exponential phase, adding cerium nitrate solution under stirring, adding ammonia water to adjust the pH value to 10 after stirring, continuing to stir for a certain period of time, centrifuging the solution to obtain a yellow precipitate, washing the yellow precipitate, drying it below 100°C, grinding it into powder, and obtaining the cerium oxide nanoparticles.

[0010] Preferably, the method specifically includes: taking 20 mL of Chlorella culture medium in the exponential growth phase, adding 0.5 M cerium nitrate solution under stirring, stirring for 30 min, adding 1 M ammonia water to adjust the pH value to 10, observing that the color of the solution gradually changes from light yellow to yellow as the ammonia water increases, continuing to stir for 1 h, centrifuging the solution to obtain a yellow precipitate, washing the yellow precipitate with acetone and water, drying it at 80 °C overnight, grinding the dried sample into powder to obtain the cerium oxide nanoparticles.

[0011] The present invention also provides an application of the cerium oxide nanoparticles described in any of the above technical solutions in the antibacterial coating of urinary catheters.

[0012] Preferably, the method includes: preparing a gel solution using the cerium oxide nanoparticles, chitosan, and genipin as raw materials, immersing the urinary catheter in the gel solution, and forming a gel coating on the surface of the urinary catheter.

[0013] Preferably, the cerium oxide nanoparticles account for 0.54% of the dry weight of the gel coating.

[0014] Preferably, the gel coating forms a plurality of pores with a diameter of 48-52 nm.

[0015] Preferably, the preparation process of the gel solution includes: mixing a 0.4 wt% chitosan solution with pH 5.0 and a 2M genipin solution at a volume ratio of 5:2, then adding a 71 mg / mL cerium oxide nanoparticle solution, and stirring thoroughly to form the hydrogel solution.

[0016] Preferably, the process of forming a gel coating on the surface of the urinary catheter includes: ultrasonically cleaning the sterile urinary catheter in anhydrous ethanol, immersing it in a gel solution for 5 minutes, removing it, immersing it in a 1M NaOH solution, letting it stand for 5 minutes, and removing it after the gel coating has solidified.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] This invention provides cerium oxide nanoparticles. By introducing Chlorella into the cerium oxide nanoparticles, not only are the cerium oxide nanoparticles endowed with biological activity, making them more suitable for use in urinary catheters, but the introduction of Chlorella also greatly improves the catalytic activity and oxygen free radical generation ability of the cerium oxide nanoparticles, thereby improving the antibacterial efficiency and bactericidal performance of the cerium oxide nanoparticles.

[0019] This invention provides a method for preparing cerium oxide nanoparticles, which is simple to operate and low in cost;

[0020] This invention also provides the application of cerium oxide nanoparticles in the antibacterial coating of urinary catheters, which has the characteristics of higher biosafety and higher antibacterial efficiency. Attached Figure Description

[0021] Figure 1 This is a TEM schematic diagram of cerium oxide nanoparticles provided in an embodiment of the present invention;

[0022] Figure 2 This is a TEM schematic diagram of the gel coating provided in an embodiment of the present invention;

[0023] Figure 3 This is a schematic diagram illustrating the change in Escherichia coli colony count with cerium oxide concentration, provided in an embodiment of the present invention.

[0024] Figure 4 This is a schematic diagram of biofilm detection on the catheter surface provided in an embodiment of the present invention. Detailed Implementation

[0025] The technical solutions in specific embodiments of the present invention will be described in detail and completely below. Obviously, the described embodiments are only some specific implementations of the overall technical solution of the present invention, and not all implementations. Based on the overall concept of the present invention, all other embodiments obtained by those skilled in the art fall within the protection scope of the present invention.

[0026] This invention provides, in one aspect, cerium oxide nanoparticles comprising cerium oxide and Chlorella vulgaris, wherein the Chlorella vulgaris is adsorbed onto the surface of the cerium oxide, and the cerium oxide contains Ce. 3+The cerium oxide nanoparticles contain 20-25% oxygen vacancies, with a relative ratio of 2.0-3.0 between oxygen vacancies and lattice oxygen. By introducing Chlorella into the cerium oxide nanoparticles, the process not only endows them with bioactivity, but also enhances their suitability for use in urinary catheters by allowing the Chlorella to adhere to the nanoparticles. Furthermore, the introduction of Chlorella significantly improves the catalytic activity and oxygen free radical generation capacity of the cerium oxide nanoparticles, thereby increasing their antibacterial efficiency and bactericidal properties. It should be noted that the reason why the introduction of Chlorella can significantly improve the catalytic activity and oxygen free radical generation capacity of the cerium oxide nanoparticles is that it acts as a reducing agent and / or oxidizing agent and end-capping agent to stabilize NPs, altering the particle size, morphology, and band gap energy of the synthesized material, which is beneficial for optimizing the morphology of the NPs. Furthermore, this technical solution selects Chlorella vulgaris instead of other algae to incorporate cerium oxide nanoparticles because Chlorella vulgaris grows rapidly and produces a large amount of reducing sulfur and nitrogen metabolites. These reducing substances produced by Chlorella vulgaris are used to reduce cerium nitrate, thereby forming biomimetic mineralized Bio-CeO2 nanoparticles. This technical solution specifically limits the Ce content in cerium oxide... 3+ The proportion is due to the higher Ce 3+ The ratio of CeO2 to CeO2 is directly related to its catalytic activity; a higher ratio generally results in a stronger ability to generate free radicals. This can be understood as the proportion of CeO2 in cerium oxide being higher than that in cerium oxide. 3+ The percentage can also be any value within the range of 21%, 22%, 23%, 24%, etc. This technical solution also specifically defines the relative ratio of oxygen vacancies to lattice oxygen in cerium oxide nanoparticles, and this ratio is higher than that of previous related studies (1.4). This also indicates that the green synthetic sample (cerium oxide nanoparticles) obtained by this invention has a higher proportion of oxygen vacancies, and correspondingly a stronger ability to generate oxygen free radicals. It is understood that this ratio can also be any value within the range of 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, etc.

[0027] In a preferred embodiment, the cerium oxide nanoparticles are spherical with a diameter of 6-7 nm and a BET of 70-80 nm. 2 / g. This embodiment specifically defines the BET of cerium oxide nanoparticles; it can be understood that it could also be 71m. 2 / g、72m 2 / g、73m 2 / g、74m 2 / g、75m 2 / g、76m 2 / g、77m 2 / g、78m 2 / g、79m 2 / g and any point value within its range. Compared to traditional cerium oxide nanoparticles that do not include Chlorella, the green cerium oxide nanoparticles containing Chlorella provided by this invention have a larger comparative area and higher surface activity.

[0028] Another aspect of the present invention provides a method for preparing cerium oxide nanoparticles according to any of the above-mentioned technical solutions, comprising: taking a culture medium of Chlorella growing in the exponential growth phase, adding cerium nitrate solution under stirring, adding ammonia water to adjust the pH value to 10 after stirring, continuing to stir for a certain period of time, centrifuging the solution to obtain a yellow precipitate, washing the yellow precipitate, drying it below 100°C, and grinding it into powder to obtain the cerium oxide nanoparticles. The introduction of Chlorella into the preparation process of cerium oxide nanoparticles results in a final cerium oxide conversion rate of 7.6%, which is a certain improvement compared to traditional methods. This is because no high-temperature calcination was performed during the entire experimental preparation process, and the active substances produced by Chlorella adhered to the Bio-CeO2 (cerium oxide nanoparticles), leading to a slight increase in its conversion rate.

[0029] In a preferred embodiment, the process specifically includes: taking 20 mL of Chlorella culture medium in the exponential growth phase, adding 0.5 M cerium nitrate solution under stirring, stirring for 30 min, adding 1 M ammonia to adjust the pH to 10, observing that the solution gradually changes from light yellow to yellow as the ammonia increases, continuing to stir for 1 h, centrifuging the solution to obtain a yellow precipitate, washing the yellow precipitate with acetone and water, drying it overnight at 80 °C, grinding the dried sample into powder to obtain the cerium oxide nanoparticles.

[0030] This invention also provides an application of the cerium oxide nanoparticles described in any of the above-mentioned technical solutions in the antibacterial coating of urinary catheters. It should be noted that, during use, a urinary catheter is inserted into the bladder via the urethra to drain urine. After insertion, a balloon near the catheter tip secures the catheter within the bladder, preventing it from easily dislodging. The drainage tube is connected to a urine bag to collect urine. To prevent infection, the sterility requirements for the urinary catheter are extremely high. Therefore, improving the antibacterial properties of the urinary catheter is of great significance. Furthermore, given the special usage method of the urinary catheter, its biosafety is also particularly important. This invention provides green cerium oxide nanoparticles containing Chlorella, which, when applied to the antibacterial coating of urinary catheters, exhibit higher biosafety and higher antibacterial efficiency.

[0031] In a preferred embodiment, the process includes: preparing a gel solution using the cerium oxide nanoparticles, chitosan, and genipin as raw materials; immersing a urinary catheter in the gel solution; and forming a gel coating on the surface of the urinary catheter. This embodiment specifically defines the preparation method of the gel coating and specifically defines the use of chitosan and genipin as raw materials in the preparation of the gel solution. Chitosan forms a layered structure, and genipin connects the layers to form a network structure, improving the stability of the gel and providing more connection sites for the cerium oxide nanoparticles, which is beneficial for the uniform distribution of the cerium oxide nanoparticles, thereby ensuring its stable and efficient antibacterial properties. Specifically, the preparation process of the gel solution includes: mixing a 0.4 wt% chitosan solution with pH 5.0 and a 2M genipin solution at a volume ratio of 5:2, adding a 71 mg / mL cerium oxide nanoparticle solution, and stirring thoroughly to form the hydrogel solution. The process of forming a gel coating on the surface of the urinary catheter includes: ultrasonically cleaning the sterile urinary catheter in anhydrous ethanol, immersing it in a gel solution for 5 minutes, removing it, immersing it in a 1M NaOH solution, letting it stand for 5 minutes, and removing it after the gel coating has solidified.

[0032] In a preferred embodiment, the cerium oxide nanoparticles account for 0.54% of the dry weight of the gel coating.

[0033] In a preferred embodiment, the gel coating forms a plurality of pores with a diameter of 48-52 nm, indicating that the gel coating has a high water content and good hydrophilicity. It is understood that the pore diameter can also be any value within the range of 49 nm, 50 nm, 51 nm, or any point within that range.

[0034] To provide a clearer and more detailed description of the cerium oxide nanoparticles, their preparation methods, and applications provided in the embodiments of the present invention, the following description will be based on specific embodiments.

[0035] Example 1

[0036] Preparation of cerium oxide nanoparticles: Take 20 mL of Chlorella culture medium in the exponential growth phase, add 0.5 M cerium nitrate solution under stirring, stir for 30 min, then add 1 M ammonia to adjust the pH to 10.0. Observe that the solution gradually changes from light yellow to yellow as the ammonia increases. After stirring for 1 h, the solution is precipitated and centrifuged. The yellow precipitate is washed three times with acetone and water to remove nitrates, ammonia and other organic impurities. Finally, dry at 80 °C overnight, grind the dried sample into powder to complete the preparation of Bio-CeO2 NPs (cerium oxide nanoparticles), and store in a dry place.

[0037] Preparation of the gel: 0.4 wt% pH 5.0 chitosan solution and 2M genipin solution were mixed at a volume ratio of 5:2. Then, 71 mg / mL cerium oxide nanoparticles were added and stirred thoroughly to form a hydrogel solution. The sterile urinary catheter was ultrasonically cleaned three times in anhydrous ethanol, then immersed in the gel solution for 5 min. After removal, it was immersed in 1M NaOH solution and allowed to stand for 5 min. After the gel coating solidified, it was removed to obtain a urinary catheter with an antibacterial coating.

[0038] Performance testing

[0039] I. Physicochemical Characterization of Cerium Oxide Nanoparticles and Hydrogels

[0040] 1. Physicochemical characterization of cerium oxide nanoparticles

[0041] TEM results: Figure 1 As shown, Bio-CeO2 is spherical with a diameter of about 6-7 nm, and most of the particles are between 9-10 nm in diameter.

[0042] XRD characterization: The XRD pattern of Bio-CeO2 is basically consistent with the standard PDF card (JCPDS card number 43-1002) of CeO2, indicating that there are few other crystalline impurities in the sample. The XRD pattern of commercial C-CeO2 also shows a similar pattern, and the lattice size of both samples is 5.4.

[0043] BET results: BET of Bio-CeO2 (73m) 2 / g) is significantly higher than C-CeO2 (30m) 2 The ratio of / g) indicates that the green-synthesized nanoparticles have a larger comparative area and higher surface activity.

[0044] XPS analysis: The Ce 3d spectrum shows that the peaks at 884.8 eV, 02.8 eV, and 888.7 eV correspond to Ce. 3+ The peak values ​​at 883.1 eV, 889.5 eV, 889 eV, 901.6 eV, 908.1 eV, and 917.3 eV correspond to Ce. 4+ Fitting analysis inferred Ce 3+ The percentage is 21%. According to previous reports, the higher Ce... 3+ The ratio of oxygen to lattice oxygen is directly related to the catalytic activity of CeO2; a higher ratio generally indicates a stronger ability to generate free radicals. The O 1s spectrum showed characteristic peaks for oxygen, originating from surface OH groups or H2O (529.5 eV), oxygen vacancies (532.4 eV), and lattice oxygen (533.8 eV). The relative ratio of oxygen vacancies to lattice oxygen was 2.5, higher than the 1.4 in previous studies, indicating that the green synthesis sample in this project had a higher proportion of oxygen vacancies, resulting in a stronger ability to generate oxygen free radicals.

[0045] Size: Most Bio-CeO2 particles are around 50nm in size, slightly larger than the average particle size of a single particle (8.9nm), indicating that some particles aggregate into clusters. However, these aggregated particles are evenly distributed, and most of them are no larger than 100nm, indicating that the particles have good suspension in the solution and are not large in size. They are expected to be evenly distributed in the coating gel.

[0046] 2. Physicochemical characterization of hydrogels

[0047] like Figure 2 As shown in the cross-section, the hydrogel contains numerous pores with a diameter of approximately 50 nm, indicating a potentially high water content and good hydrophilicity. CeO2 particles are uniformly distributed within the gel, with Ce accounting for 0.54% (w / w) of the gel's dry weight.

[0048] II. Enzyme-mimicking activity of cerium oxide nanoparticles

[0049] 1. The activity of the bio-CeO2 mimic enzyme was determined using TMB and OPD, common substrates for oxidases and peroxidases: their oxidation products exhibit specific absorption peaks at 652 and 450 nm, respectively. Therefore, the activity of the mimic enzyme can be quantitatively tested based on its absorbance intensity. Bio-CeO2 was added alone... 2 The characteristic absorption peak intensities of the latter two substrates both increased, indicating that cerium oxide itself can directly catalyze the oxidation of both substrates, i.e., it possesses oxidase-like activity. The substrate oxidation ability of Bio-CeO2 is significantly enhanced with the addition of H2O2, far exceeding that of Bio-CeO2 or H2O2 alone. Previous studies have shown that cerium oxide possesses peroxidase-like activity, capable of decomposing H2O2 into hydroxyl radicals HO·. Hydroxyl radicals have higher oxidizing power than most reactive oxygen species (ROS) such as O2·, thus exhibiting stronger substrate oxidation capabilities. Commercial C-CeO2 also possesses oxidase-like and peroxidase-like functions, but its activities are 26% and 53% lower than Bio-CeO2, respectively. This may be because Bio-CeO2 has a smaller size, higher specific surface area, and more surface defects, thus resulting in a stronger catalytic ability to produce ROS.

[0050] 2. The generation of reactive oxygen species was detected by the fluorescent probe o-phenylenediamine (TA): After the addition of Bio-CeO2, the fluorescence of TA was greatly enhanced, proving its ability to generate reactive oxygen species.

[0051] III. Antibacterial Activity of Cerium Oxide Nanoparticles

[0052] 1. Colony counting method

[0053] Compared to the control group, only 10 μg / mL of Bio-CeO2 could inhibit 90% of E. coli and 80% of Pae, demonstrating a significant antibacterial effect. Using 0.05 mM H2O2, the antibacterial effect of Bio-CeO2 became even more pronounced after the addition of H2O2; the survival rate of both E. coli and Pae decreased to below 5%, indicating that the hydroxyl radicals generated after the reaction of cerium oxide with H2O2 have a stronger bactericidal effect. Figure 3 As shown, the number of Escherichia coli colonies gradually decreased with increasing cerium oxide concentration.

[0054] 2. Group sports

[0055] Compared to the control group, in the absence of H2O2, the migration distance of both E. coli and Pae bacteria on CeO2-coated plates was reduced. Bio-CeO2 particles showed better antibacterial effects than C-CeO2, and the effect was even more pronounced with the addition of H2O2, reducing the bacterial count by up to 25%.

[0056] 3. Inhibition of biomembranes

[0057] The integrity of the biofilm was significantly disrupted after the addition of 200 μg / mL Bio-CeO2 or C-CeO2, with the effect being even more pronounced in the presence of 0.05 mM H2O2. Figure 4 As shown, the coated catheters have less biofilm on their surface than the uncoated catheters.

[0058] 4. Fluorescent staining

[0059] AO / EB staining results showed that when *E. coli* and *Pseudomonas aeruginosa* were stained with fluorescent dyes, almost no bacteria died without Bio-CeO2 or H2O2. With the addition of Bio-CeO2, the percentage of dead cells gradually increased, and the red fluorescence became dominant. With the addition of H2O2, the number of dead cells increased significantly; only a very small number of *E. coli* cells remained viable, and *Pseudomonas aeruginosa* cells showed virtually no green fluorescence. Therefore, under the condition of co-treatment with Bio-CeO2 and H2O2, the number of dead cells in both bacteria increased significantly, and the red fluorescence increased markedly.

[0060] 5. Hydrogel coating has antibacterial properties.

[0061] The absorbance of the coated catheter surface was lower than that of the uncoated catheter, demonstrating a significant inhibitory effect on biofilm formation. Furthermore, the amount of biofilm on the coated catheter surface was further reduced upon the addition of H₂O₂. That is, Bio-CeO₂ + H₂O₂ > Bio-CeO₂ > Uncoated.

Claims

1. A method for preparing cerium oxide nanoparticles, characterized in that, include: Take 20 mL of Chlorella culture medium in the exponential growth phase, add 0.5 M cerium nitrate solution under stirring, stir for 30 min, then add 1 M ammonia to adjust the pH to 10. Observe that the color of the solution gradually changes from light yellow to yellow as the ammonia increases. After stirring for 1 h, the solution is precipitated and centrifuged to obtain a yellow precipitate. Wash the yellow precipitate with acetone and water, dry it at 80℃ overnight, and grind the dried sample into powder to obtain cerium oxide nanoparticles. The cerium oxide nanoparticles comprise cerium oxide and Chlorella vulgaris, with the Chlorella vulgaris adsorbed onto the surface of the cerium oxide. The cerium oxide contains Ce. 3+ The proportion is 20-25%, and the relative ratio of oxygen vacancies to lattice oxygen in the cerium oxide nanoparticles is 2.0-3.

0.

2. The method for preparing cerium oxide nanoparticles according to claim 1, characterized in that, The cerium oxide nanoparticles are spherical with a diameter of 6-7 nm and a BET of 70-80 μm. 2 / g.

3. The application of the cerium oxide nanoparticles prepared by the method according to any one of claims 1-2 in the antibacterial coating of urinary catheters.

4. The application of cerium oxide nanoparticles according to claim 3 in the antibacterial coating of urinary catheters, characterized in that, include: A gel solution was prepared using the cerium oxide nanoparticles, chitosan, and genipin as raw materials. The catheter was then immersed in the gel solution to form a gel coating on the surface of the catheter.

5. The application of cerium oxide nanoparticles according to claim 4 in the antibacterial coating of urinary catheters, characterized in that, The cerium oxide nanoparticles account for 0.54% of the dry weight of the gel coating.

6. The application of cerium oxide nanoparticles according to claim 4 in the antibacterial coating of urinary catheters, characterized in that, The gel coating forms several pores with a diameter of 48-52 nm.

7. The application of cerium oxide nanoparticles according to claim 4 in the antibacterial coating of urinary catheters, characterized in that, The preparation process of the gel solution includes: mixing a 0.4 wt% chitosan solution with pH 5.0 and a 2 M genipin solution at a volume ratio of 5:2, then adding a 71 mg / mL cerium oxide nanoparticle solution and stirring thoroughly to form the gel solution.

8. The application of cerium oxide nanoparticles according to claim 4 in the antibacterial coating of urinary catheters, characterized in that, The process of forming a gel coating on the surface of the urinary catheter includes: ultrasonically cleaning the sterile urinary catheter in anhydrous ethanol, immersing it in a gel solution for 5 minutes, removing it, immersing it in a 1 M NaOH solution, letting it stand for 5 minutes, and removing it after the gel coating has solidified.

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